Exonuclease fusing ntpase domain and application thereof

Abstract

The application discloses an exonuclease fusing an NTPase domain and application thereof, and belongs to the technical field of bioengineering. The exonuclease is a Ppl protein, which comprises a PHP domain at the N terminal and an NTPase domain at the C terminal, wherein the PHP domain has 3'-5' ssDNA exonuclease activity. The NTPase domain of the application regulates the exonuclease activity of the PHP domain through conformational change. Under the condition of high NTP concentration, the NTPase domain inhibits the activity of the PHP domain; when the NTP concentration decreases, the inhibition is released, and the exonuclease activity is significantly activated. The activated PHP domain shows specific cleavage activity to DNA substrates with 3'-hydroxyl overhang. The unique 'NTP concentration sensing and enzyme activity regulation' characteristics make it a core module for developing biological sensors, gene circuits and controllable nucleic acid tools.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, and more specifically, to a nuclease fused with an NTPase domain and its applications. Background Technology

[0002] Exonucleases play crucial roles in DNA metabolism regulation, genome stability maintenance, and the execution of various cellular processes. With the continuous development of structural biology, genomics, and bioinformatics, it has become increasingly clear that a class of multi-domain nucleic acid processing proteins, highly integrated with multiple functional domains, are widely present in microbial genomes. These proteins couple energy-dependent regulatory modules with nucleic acid catalytic domains, enabling more complex and precise regulation of their nucleic acid degradation activities. For example, the P-loop NTPase domain is a conserved protein folding mechanism capable of binding and hydrolyzing ATP or GTP. It drives conformational changes to achieve energy state sensing, activity regulation, or complex assembly, and is an important molecular switch module widely present in prokaryotes and eukaryotes (Walker, JE et al., 1982, EMBO J., 1, 945-951; Leipe, DD et al., 2002, Journal of Molecular Biology, 317, 41-72). On the other hand, the PHP (polymerase and histidinol phosphatase) domain and related metal-dependent nuclease domains play a core role in nucleic acid cleavage and repair. Their catalytic centers depend on divalent metal ions and can mediate specific or non-specific hydrolysis of single-stranded or double-stranded nucleic acids (Aravind, L. et al., 1998, Nucleic Acids Research, 26, 4205–4213).

[0003] In recent years, genome proximity analysis, structural prediction, and evolutionary analysis have revealed a common type of single-domain protein in a large number of bacterial genomes, consisting of a combination of a P-loop NTPase domain and a PHP-like exonuclease domain (Iyer, LM et al., 2004, Journal of Structural Biology, 146, 11-31; Makarova, KS et al., 2019, Nature Reviews Microbiology, 17, 67-88). This structural integration differs from classic two-component systems. For example, the Gabija system consists of two independent proteins, including a sensor protein responsible for signal recognition and an effector protein that performs nucleic acid cleavage, and its regulation depends on the activation cascade between proteins (Gao, L. et al., 2020, Science, 369, 1077-1084). The Hachiman system also consists of two proteins, and the initiation of its nuclease function depends on the sensing and transmission of specific intracellular signals (Tuck, OT et al., 2024, Cell, 187, 6914-6928). These two-component systems require precise interactions between multiple proteins in terms of both structure and regulation, resulting in a relatively complex overall network. Summary of the Invention

[0004] To overcome the shortcomings of existing exonucleases in the prior art, such as lack of metabolic state response capability and inability to regulate enzyme activity based on NTP concentration, this invention provides an exonuclease fused with an NTPase domain. Another object of the present invention is to provide a polynucleotide; Another object of the present invention is to provide a carrier; Another object of the present invention is to provide a host cell; Another object of the present invention is to provide a method for regulating the activity of exonucleases; Another objective of this invention is to provide an application of an exonuclease fused with an NTPase domain in the preparation of biosensors; Another objective of this invention is to provide an exonuclease fused with an NTPase domain for use as a controllable nucleic acid editing tool in molecular biology.

[0005] To solve the above-mentioned technical problems, the technical solution of the present invention is as follows: An exonuclease fused with an NTPase domain, the exonuclease comprising an N-terminal PHP domain and a C-terminal NTPase domain; the C-terminal NTPase domain can inhibit the 3'-5' ssDNA exonuclease activity of the N-terminal PHP domain through a conformational change, and the strength of this inhibition is positively correlated with the concentration of nucleoside triphosphates. The exonuclease is a Ppl protein, composed of... ppl The gene encodes an amino acid sequence as shown in SEQ ID NO 1; or a protein homologous to the sequence shown in SEQ ID NO 1; or a functional fragment or derivative thereof; wherein the homologous protein, functional fragment or derivative has the NTP concentration-regulated exonuclease activity; SEQ ID NO 1: MVGSRWYKFDFHNHTPASHDYKIPDISPREWLLAYMKQHVDCVVISDHNSGAWVDVLKGELENMSRDASTGDLPEFRPLTLFPGVELTATGNVHILAVLHTHSTSADVE RLLAQCNNNSPIPSEVPNHQLVLQLGPAGIISNIRRNPKAVCILAHIDAAKGVLSLTNQAELTAAFQESPHAVEIRHRVEDITDGTRRRLIDNLPWLRGSDAHHPEQAGVRT CWLKMSSPDFDGLRHALLDPENCVLFDQLPPEEPASYLRSLKFRTRHCHPVGQDSASVEFSPFYNAVIGSRGSGKSTLIESIRLAMRKTEGLTATQGSKLDQFIRTGMEAD SFIECIFHKEGTDFRLSWRPDSKHELHIFSDGEWMPDSHWSADRFPLSIYSQKMLYELASDTGAFLRVCDESPVVNKRAWKERWDQLEREYLNEQITLRGLRARQGSADSLR GELSDAERAVSQLQSSAYYPVCRQLALARNELSAATLPLEHFERRIAAIQALAEEPLQRSDIPPEPSGLLMAFMARLSSVQQQYDQRLNTLLAEYAAELAGIRREQSFIAL RTAVSDQETNVESEAVSLRARGLNPDVLNELMARCESLKNELRNYDGLDGAISASVARSEQLLAEMRAHRMALTDNRKAFLSSLSLSALEIKILPLCAPYEDVISGYQTVTG ISNFAERIYDNSDGSGLLSDFISERPFSPLPAATEKKYRALDELKALHHSIRLDNSEAGAGLHGSFRNRLRSLNDQQLDALQCWYPDDGIHIRYQTPGGQMEDIAFASPGQK GASMLQFLLSYGTDPLLLDQPEDDLDCLMLMSVIPAIMSNKKRRQLIIVSHSAPIVVNGDAEYVISMQHDRTGLYPGLCGALQEAPMKALICRQMEGGEKAFRSRYERILS The present invention relates to an exonuclease fused with an NTPase domain, comprising an N-terminal PHP domain and a C-terminal NTPase domain, and homologous, mutated, or modified genes and proteins thereof.

[0006] The amino acid sequence of the enzyme described above is shown in SEQ ID NO 1. Its PHP domain possesses 3'-5' ssDNA exonuclease activity, and its NTPase domain can regulate this activity through NTP concentration-dependent conformational changes. Alternatively, it may be a homologous protein with high protein sequence homology (sequence identity not less than 70%, 80%, 90%, or 95%) to the sequence shown in SEQ ID NO 1, and performing the same or similar regulatory functions in vivo; or a derivative protein by substituting, deleting, and / or adding one or more amino acids to the sequence shown in SEQ ID NO 1, while retaining the NTP concentration-regulated exonuclease activity. The nucleotide sequence of the enzyme's encoding gene is preferably shown in SEQ ID NO 2.

[0007] Furthermore, the functional fragment is a truncated protein containing the PHP domain and the NTPase domain.

[0008] Furthermore, the derivative is a protein that has undergone substitution, deletion, and / or addition of one or more amino acids and has the NTP concentration-regulated exonuclease activity.

[0009] A polynucleotide encoding the exonuclease.

[0010] Further, the nucleotide sequence of the polynucleotide is as shown in SEQ ID NO 2; or a variant thereof having sequence homology with the sequence shown in SEQ ID NO 2; or a degenerate sequence thereof;

[0011] A vector comprising the aforementioned polynucleotide. The vector contains the nucleotide sequence shown in SEQ ID NO.2, which enables the expression of the NTP-regulated exonuclease in prokaryotic or eukaryotic host cells.

[0012] A host cell containing the aforementioned vector, or having the aforementioned polynucleotide integrated into its genome. This host cell can be used in basic research, biomanufacturing, or as an industrial strain resistant to phage contamination.

[0013] A method for regulating the activity of exonucleases involves altering the concentration of nucleoside triphosphates (NTPs) in the reaction system. This regulation manifests as activity inhibition at high NTP concentrations and reactivation at low NTP concentrations. This method greatly facilitates the controllable operation of in vitro biochemical reactions.

[0014] Application of the described exonuclease fused with an NTPase domain in the preparation of biosensors.

[0015] Preferably, the application is in the controlled cleavage of nucleic acids in response to cellular metabolic states. Specifically, the activity of the enzyme is inhibited at high NTP concentrations (such as during physiological proliferation); when the NTP concentration decreases (such as during stress or after phage hijacking of cellular resources), its exonuclease activity is specifically activated, thereby performing the cleavage function. This characteristic enables it to serve as a precision sensor for the intracellular nucleotide metabolic state.

[0016] Application of the described exonuclease fused with an NTPase domain as a controllable nucleic acid editing tool in molecular biology.

[0017] Preferably, it is used in the specific cleavage of DNA substrates with 3'-hydroxyl overhangs. This specificity ensures its ability to manipulate precisely in complex genomic contexts.

[0018] The applications include, but are not limited to, life science research, molecular diagnostics, biotechnology development, and industrial fermentation. Specifically, they include: serving as a controllable auxiliary tool in gene editing to optimize editing efficiency or reduce off-target effects; serving as a tool for studying DNA repair pathways in cells under energy stress in DNA damage repair research; serving as a core element in constructing NTP concentration-responsive gene circuits in synthetic biology; serving as a switching component for signal amplification or control in the development of novel molecular diagnostic platforms; and serving as a defense element to enhance the phage resistance of engineered strains in industrial fermentation.

[0019] This invention provides a novel fusion exonuclease with precisely regulated activity. The enzyme's activity is precisely regulated by the concentration of an endogenous cellular signal—nucleoside triphosphate (NTP). The exonuclease activity of its PHP domain is inhibited by its own NTPase domain and significantly activated when NTP concentration decreases. The activated PHP domain exhibits specific cleavage activity against DNA substrates with 3'-hydroxyl overhangs.

[0020] This invention provides the gene sequence and protein information of an exonuclease fused with an NTPase domain, revealing the activity regulation mechanism of this enzyme in response to NTP concentration. This lays the foundation for the development of novel molecular switch tool enzymes, which can be widely applied in fields such as gene editing system optimization, DNA damage repair research, molecular diagnostic technology development, and precise regulation of synthetic biological elements.

[0021] Compared to the aforementioned multi-protein complex systems, the NTPase-PHP fusion protein of interest in this invention integrates the energy-sensing module and the catalytic module directly into the same protein molecule, forming a highly compact, integrated structure. The NTPase module can directly sense the concentration of nucleoside triphosphates (NTPs) in the cell and drive conformational changes, thereby regulating the accessibility or catalytic state of the nuclease domains and achieving direct coupling between energy metabolism levels and nucleic acid processing activity. Structural prediction studies further demonstrate that these multi-domain proteins exhibit a tightly connected modular layout in their three-dimensional conformation, significantly improving the efficiency of energy conversion and catalytic activity switching.

[0022] Thanks to their unique domain synergistic mechanisms and energy-dependent catalytic regulation characteristics, NTPase-nuclease fusion proteins have shown broad application potential in various bioengineering scenarios: in synthetic biology, they can serve as metabolic state-responsive regulatory modules; in industrial strain modification, they can enhance genome homeostasis and fermentation robustness; and in biosensor construction, they hold promise for enabling programmable nucleic acid responses based on NTP levels, energy states, or metabolic signals. Therefore, these multi-domain fusion enzymes represent a novel class of biological functional modules with unique regulatory logic and significant application prospects, providing valuable resources for the development of novel biotechnological tools.

[0023] Compared with the prior art, the beneficial effects of the technical solution of the present invention are: This invention elucidates and verifies for the first time at the molecular level a self-regulating exonuclease encoded by a single gene and containing an NTPase domain. The core advantage of this enzyme lies in its ingenious "built-in switch" mechanism: its C-terminal NTPase domain can directly sense changes in the concentration of key intracellular metabolites (NTPs) and, through conformational rearrangement, precisely regulate the exonuclease activity of its N-terminal PHP domain. This characteristic of "high NTP inhibition and low NTP activation" makes it a natural biosensor and molecular switch capable of directly reading the cellular metabolic state.

[0024] Compared to traditional controllable enzyme tools that require the addition of small molecule inducers or physical signals (such as light or heat), the enzyme described in this invention utilizes the fundamental and dynamically changing physiological signal of intracellular nucleotide metabolism as a regulatory switch, offering advantages such as no exogenous intervention, rapid response, and natural coupling with cellular physiological states. This unique mechanism not only provides a novel core tool for constructing intelligent feedback regulatory loops in living cells and studying cellular stress responses, but also opens new pathways for developing novel molecular diagnostic technologies based on metabolic states and constructing more robust industrial strains. This invention significantly expands the application boundaries of microbial defense system components in synthetic biology and biotechnology. Attached Figure Description

[0025] Figure 1 This is a schematic diagram illustrating the construction of plasmid pWHU4601; Figure 2 Electrophoresis image of fusion exonuclease in in vitro DNA cleavage reaction; Figure 3 A diagram showing the detection of NTP hydrolysis reactions involving fused exonucleases; Figure 4 To detect DNA cleavage reactions under different nucleoside triphosphate conditions; Figure 5 Graphs showing the DNA cleavage reaction of individual PHP domain proteins under different nucleoside triphosphate conditions; Figure 6 This image shows the detection of DNA damage induced by fusion exonucleases in cells; A represents cell morphology, and B represents TUNEL flow cytometry. Detailed Implementation

[0026] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0027] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0028] Example 1 Construction of plasmid pWHU4601 containing a fusion exonuclease gene fragment Synthetic containing ppl The nucleotide sequences of the gene and its promoter are shown in SEQ ID NO 2. Using primers 5'-GGATCCTCTACGCCGGA-3' and 5'-GGATCCACAGGACGGGTGT-3', plasmid backbone nucleic acid fragment 1 was obtained by PCR using plasmid pACYC184 as a template; using primers 5'-CCCGTCCTGTGGATCCAAATTATCATCCTTGATGGA-3' and 5'-CCGGCGTAGAGGATCCTCAGCTAAGAATACGCTCAT-3', ... ppl Gene sequence as template PCR to obtain containing ppl Nucleic acid fragment 2 of the gene; fragments 1 and 2 were homologously recombinated using the Gibson splicing cloning kit to obtain the complete circular plasmid pWHU4601 ( Figure 1 ), which includes from Escherichia coli ( E. coli NCTC8620 ppl Genes and primitive promoters.

[0029]

[0030] Example 2 The fusion exonuclease cleaves single-stranded DNA substrates under in vitro conditions. Using plasmid pWHU4601 as a template, nucleic acid fragment 1 was obtained by PCR with primers 5'-CTTTAAGAAGGAGATATACCATGGTAGGTTCGCGCTGGTAT-3' and 5'-TCAGTGGTGGTGGTGGTGGTGCTCGAGGCTAAGAAT ACGCTCATAGC-3'. Plasmid backbone nucleic acid fragment 2 was obtained by PCR with primers 5'-CTCGAGCACCACCACCACCACCACCACCAC-3' and 5'-GGTATATCTCCTTCTTAAAG-3' and plasmid pET28a as a template. The above fragments 1 and 2 were homologously recombined using the Gibson splicing cloning kit to obtain the complete circular plasmid pWHU4609, which was used to express and purify the exonuclease fused with the NTPase domain. Based on this, point mutation PCR was performed on pWHU4609. The mutant control was obtained by replacing the amino acids at four key sites of the fusion exonuclease, namely D10, H12, H14 and H48, with alanine (A).

[0031] Then pWHU4609 is converted into C43 (DE3) to obtain C43 (DE3) (pWHU4609).

[0032] The bacteria were cultured overnight at 37°C in LB medium containing 50 μg / mL kanamycin. The next day, they were transferred at a 1:100 volume ratio to LB medium containing 50 μg / mL kanamycin and cultured at 37°C until the OD600 reached 0.8. The temperature was then lowered to 16°C, and IPTG was added to a final concentration of 0.4 mM. The culture was continued for 16 hours. The bacterial cells were collected, homogenized using a JNBIO homogenizer, and then centrifuged at 4°C (15000×g, 1 hour) to remove cell debris. The supernatant was filtered through a 0.22 μm filter membrane and incubated with a nickel column. Elution was performed using imidazole solutions of varying concentrations. The target protein filtrate was then collected and further purified using a molecular sieve (Superdex 200 gel filtration column, GE Healthcare). Finally, the filtrate was flash-frozen in liquid nitrogen and stored at -80°C until next use. The purified fusion exonuclease was Ppl protein, and its amino acid sequence is shown in SEQ ID NO: 1. SEQ ID NO 1:MVGSRWYKFDFHNHTPASHDYKIPDISPREWLLAYMKQHVDCVVISDHNSGAWVDVLKGELENMSRDASTGDLPEFRPLTLFPGVELTATGNVHILAVLHTHSTSADVERLLAQCNNNSPIPSEVPNHQLVLQLGPAGIISNIRRNPKAVCILAHIDAAKGVLSLTNQAELTAAFQESPHAVEIRHRVEDITDGTRRRLIDNLPWLRGSDAHHPEQAGVRTCWLKMSSPDFDGLRHALLDPENCVLFDQLPPEEPASYLRSLKFRTRHCHPVGQDSASVEFSPFYNAVIGSRGSGKSTLIESIRLAMRKTEGLTATQGSKLDQFIRTGMEADSFIECIFHKEGTDFRLSWRPDSKHELHIFSDGEWMPDSHWSADRFPLSIYSQKMLYELASDTGAFLRVCDESPVVNKRAWKERWDQLEREYLNEQITLRGLRARQGSADSLRGELSDAERAVSQLQSSAYYPVCRQLALARNELSAATLPLEHFERRIAAIQALAEEPLQRSDIPPEPSGLLMAFMARLSSVQQQYDQRLNTLLAEYAAELAGIRREQSFIALRTAVSDQETNVESEAVSLRARGLNPDVLNELMARCESLKNELRNYDGLDGAISASVARSEQLLAEMRAHRMALTDNRKAFLSSLSLSALEIKILPLCAPYEDVISGYQTVTGISNFAERIYDNSDGSGLLSDFISERPFSPLPAATEKKYRALDELKALHHSIRLDNSEAGAGLHGSFRNRLRSLNDQQLDALQCWYPDDGIHIRYQTPGGQMEDIAFASPGQKGASMLQFLLSYGTDPLLLDQPEDDLDCLMLSMSVIPAIMSNKKRRQLIIVSHSAPIVVNGDAEYVISMQHDRTGLYPGLCGALQEAPMKALICRQMEGGEKAFRSRYERILS The in vitro nucleic acid cleavage reaction of the fused exonuclease protein was as follows: 1 μmol of purified protein was mixed with 1 μmol of DNA substrate in a reaction buffer (20 mM Tris-acetic acid, 50 mM potassium acetate, 10 mM magnesium acetate, 100 µg / ml recombinant albumin, pH 7.9) and incubated at 37°C for 1 hour. Four mutant proteins were used as controls and cleavage experiments were also performed. The reaction product was terminated by adding 6× DNA loading buffer containing SDS. Separation was performed using a 20% non-denaturing polyacrylamide (PAGE) gel with 0.5xTBE buffer and a voltage of 240 V for 45 min. After electrophoresis, the results were observed using a chemical imaging system (Bio-Rad) and the images were processed using Image Lab software.

[0033] Example 3 The fusion exonuclease hydrolyzes NTPs under in vitro conditions. To detect the ability of the fusion exonuclease to hydrolyze NTPs in vitro, the purified protein was reacted with different NTP substrates (ATP, GTP, CTP, UTP) as follows: 1 μmol of purified protein and 1 μmol of NTP substrate were mixed separately in reaction buffer (20 mM Tris-acetic acid, 50 mM potassium acetate, 10 mM magnesium acetate, 100 µg / ml recombinant albumin, pH 7.9) and incubated at 37°C for 30 minutes. After the reaction, ultrapure water was added to bring the reaction volume to 200 μL. The concentration of free phosphate ions produced by the hydrolysis of NTPs was determined using a Malachite Green Phosphate Detection Kit, and the NTP hydrolase activity was calculated using a formula.

[0034] Example 4 The regulatory role of NTP on the exonuclease activity of Ppl fusion protein To investigate the effect of NTP on the exonuclease activity of the Ppl fusion protein, 3 mM NTP or AMP-PNP was added to the in vitro nucleic acid cleavage reaction of the fusion exonuclease protein. The reaction conditions were as follows: 1 μmol of purified protein was mixed with 1 μmol of DNA substrate in reaction buffer (20 mM Tris-acetic acid, 50 mM potassium acetate, 10 mM magnesium acetate, 100 µg / ml recombinant albumin, pH 7.9), and 3 mM NTP or AMP-PNP was added. The mixture was incubated at 37°C for 1 hour. The reaction product was terminated by adding 6× DNA loading buffer containing SDS, and the product was separated using a 20% non-denaturing polyacrylamide (PAGE) gel. The results are shown below. Figure 4 As shown.

[0035] (2) Using plasmid pWHU4601 as a template, PCR was performed with primers 5'-AGGGGCCCCTGGGATCCATGGTAGGTTCGCGCTGGTAT-3' and 5'-GTCACGATGCGGCCGCTTCACTCCGGAGGGAGCTGATCA A-3' to obtain nucleic acid fragment 1. Using primers 5'-TGAAGCGGCCGCATCGTGA-3' and 5'-CATGGAT CCCAGGGGCCCCT-3', and plasmid pGEX-6P-1 as a template, PCR was performed to obtain plasmid backbone nucleic acid fragment 2. The above fragments 1 and 2 were homologously recombined using the Gibson splicing cloning kit to obtain the complete circular plasmid pWHU4617, which contains only a single exonuclease domain. pWHU4617 was then transformed into BL21 (DE3) to obtain BL21 (DE3) (pWHU4617), and the exonuclease domain protein was expressed and purified in vitro. The specific experimental procedures are described in Example 3.

[0036] To further verify the functional role of the C-terminal NTPase domain in the regulatory mechanism of this invention, a control protein containing only the PHP domain was constructed. Its nucleotide sequence SEQ ID NO 3 and amino acid sequence SEQ ID NO 4 are as follows: SEQ ID NO 3:atggtaggttcgcgctggtataaatttgattttcataaccatactccggcttcgcatgattacaaaattcctgacatcagccccagagagtggcttctggcttatatgaaacagcatgtcgattgtgttgtaatcagcgatcataacagcggagcctgggtcgacgtgttgaagggtgagctggagaatatgtcccgggacgccagcaccggcgacctgccggaatttcggccactgacactctttccgggggttgaactgacagcgaccggtaacgtacatattctggctgtgctgcacacgcacagtacaagtgccgatgtggaaaggcttctggcccagtgcaataataatagccccattccgagtgaagtccctaaccatcagctcgttcttcaactgggccccgccggcatcatcagtaatatccgccgtaatccgaaggctgtttgtattcttgcgcacattgatgcagccaaaggtgtcttaagtctgactaatcaggcagagctcaccgcagcctttcaggaaagtccccatgccgttgagattcgacaccgggtggaggatatcaccgacggaacccgccggcggctgattgataatttaccgtggctacggggctctgatgcgcaccatcctgaacaagccggcgtgcgaacctgctggctgaaaatgtcatcccctgattttgacggactcaggcatgcactgctcgatccggaaaactgtgtgctgtttgatcagctccctccggag SEQ ID NO 4: MVGSRWYKFDFHNHTPASHDYKIPDISPREWLLAYMKQHVDCVVISDHNSGAWVDVLKGELENMSRDASTGDLPEFRPLTLFPGVELTATGNVHILAVLHTHSTSADVERLLAQCNNNSPIPSEV PNHQLVLQLGPAGIISNIRRNPKAVCILAHIDAAKGVLSLTNQAELTAAFQESPHAVEIRHRVEDITDGTRRRLIDNLPWLRGSDAHHPEQAGVRTCWLKMSSPDFDGLRHALLDPENCVLFDQLPPE The in vitro nucleic acid cleavage reaction of proteins with isolated exonuclease domains is as follows: 1 μmol of purified protein was mixed with 1 μmol of DNA substrate in a reaction buffer (20 mM Tris-acetic acid, 50 mM potassium acetate, 10 mM magnesium acetate, 100 µg / ml recombinant albumin, pH 7.9), and 3 mM NTP or AMP-PNP was added. The mixture was incubated at 37°C for 1 hour. The reaction product was terminated by adding 6× DNA loading buffer containing SDS. Separation was performed using a 20% non-denaturing polyacrylamide (PAGE) gel. The results are shown below. Figure 5 As shown.

[0037] Example 5 The fusion exonuclease cuts DNA under in vivo conditions. Using the T7 phage genome as a template, PCR was performed with primers 5'-TTTGGGCTAGCAGGAGATGAACGAAAGACACTTAA-3' and 5'-ATCCGCCAAAACAGCCCTATAGTTTTATGCCTTTG-3' to obtain nucleic acid fragment 1. Using primers 5'-GGCTGTTTTGGCGGAT-3' and 5'-CATCTCCTGCTAGCCCAAA-3', and plasmid pBAD24 as a template, PCR was performed to obtain plasmid backbone nucleic acid fragment 2. The above fragments 1 and 2 were homologously recombinated using the Gibson splicing cloning kit to obtain the complete circular plasmid pWHU4618, which was used to express the phage protein Gp5.3 and trigger the exonuclease to function in vivo.

[0038] To detect DNA cleavage by fusion exonucleases under in vivo conditions, pWHU4601 and pWHU4618 expressing Gp5.3 (a protein derived from T7 phage) and its derivatives were co-transformed into *E. coli* MG1655. The transformants were cultured overnight at 37°C in LB medium containing 50 µg / mL ampicillin and 50 µg / mL chloramphenicol. The overnight cultured strain was then subcultured at a 1% inoculum and cultured at 37°C (220 rpm) for 2–3 h until the logarithmic growth phase (OD600 = 0.4). The culture was then divided into two fractions, with or without 0.2% L-arabinose to induce Gp5.3 and its derivative expression. One fraction of the sample was directly observed for cell morphology under a microscope, while the remaining sample was treated with a one-step TUNEL apoptosis detection kit (green fluorescence). The treated samples were then analyzed for bacterial DNA damage using flow cytometry.

[0039] Analysis and Explanation like Figure 2 As shown, compared with the blank control, the purified Ppl fusion protein can effectively degrade single-stranded DNA substrates, exhibiting the typical gradient step degradation bands of exonucleases, clearly demonstrating that the Ppl fusion exonuclease of this application can perform single-base cleavage of substrate DNA under in vitro conditions.

[0040] Figure 3 The results showed that after the addition of the complete Ppl fusion protein, significant detection signals appeared in all groups (ATP, GTP, CTP, and UTP), confirming that the Ppl fusion protein can effectively hydrolyze the above four NTPs and release inorganic phosphate, demonstrating a clear NTPase catalytic ability. Furthermore, the different activities of the Ppl fusion protein on different NTP substrates further prove that this NTPase activity is an inherent function of the Ppl fusion protein itself.

[0041] Figure 4 The results showed that the exonuclease activity of the Ppl fusion protein decreased significantly with increasing ATP concentration, and GTP, CTP and UTP were also inhibited, demonstrating dependence on NTP concentration and type. Figure 5 The results show that after removing the NTPase domain, the exonuclease activity of the PHP domain alone is no longer affected by NTPs such as ATP. These results indicate that the inhibitory effect of NTPs on Ppl exonuclease activity does not act directly on the PHP domain, but rather depends on the intact structure of the Ppl fusion protein. The Ppl fusion protein exhibits a unique regulatory pattern of being inhibited with increasing NTP concentration and activated with decreasing NTP concentration.

[0042] Under physiological conditions in vivo, the Ppl fusion protein can respond to changes in intracellular NTP concentration, activate and achieve DNA cleavage, and possess in vivo functional activity. Figure 6 Cell morphology observation experiments demonstrated that after Gp5.3 expression was induced to consume intracellular NTPs, the bacterial cells exhibited a filamentous phenotype caused by DNA damage. Figure 6 B TUNEL flow cytometry assays demonstrated that the positive rate of DNA damage in the experimental group was significantly higher than that in the control group, clearly confirming that the reduction of intracellular NTP levels can relieve the inhibitory state of Ppl, enabling it to be activated and cleave DNA in vivo, proving that the Ppl fusion protein of this application can function in living cells.

[0043] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. Those skilled in the art can make other variations or modifications based on the above description. It is neither necessary nor possible to exhaustively describe all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A nuclease exonuclease fused with an NTPase domain, characterized in that, The exonuclease comprises an N-terminal PHP domain and a C-terminal NTPase domain; the C-terminal NTPase domain can inhibit the 3'-5' ssDNA exonuclease activity of the N-terminal PHP domain through conformational changes, and the strength of this inhibition is positively correlated with the concentration of nucleoside triphosphate. The exonuclease is a Ppl protein, composed of... ppl The gene encodes an amino acid sequence as shown in SEQ ID NO: 1; or a protein homologous to the sequence shown in SEQ ID NO: 1; or a functional fragment or derivative thereof; wherein the homologous protein, functional fragment or derivative has the NTP concentration-regulated exonuclease activity.

2. The exonuclease fused with an NTPase domain according to claim 1, characterized in that, The functional fragment is a truncated protein containing the PHP domain and the NTPase domain.

3. The exonuclease fused with an NTPase domain according to claim 1, characterized in that, The derivative is a protein that has undergone substitution, deletion, and / or addition of one or more amino acids and has the NTP concentration-regulated exonuclease activity.

4. A polynucleotide, characterized in that, The polynucleotide encodes an exonuclease fused with an NTPase domain as described in any one of claims 1 to 3.

5. The polynucleotide according to claim 4, characterized in that, The nucleotide sequence of the polynucleotide is as shown in SEQ ID NO: 2; or a variant thereof that is sequence homologous to the sequence shown in SEQ ID NO: 2; or a degenerate sequence thereof.

6. A carrier, characterized in that, The vector comprises the polynucleotide as described in claim 4 or 5.

7. A host cell, characterized in that, The host cell contains the vector of claim 6, or its genome is integrated with the polynucleotide of claim 4 or 5.

8. A method for regulating the activity of exonucleases, characterized in that, The activity of the exonuclease fused with the NTPase domain as described in any one of claims 1 to 3 can be regulated by changing the concentration of nucleoside triphosphate in the reaction system.

9. The application of the exonuclease fused with an NTPase domain as described in any one of claims 1 to 3 in the preparation of biosensors.

10. The application of the exonuclease fused with an NTPase domain as described in any one of claims 1 to 3 as a controllable nucleic acid editing tool in molecular biology.

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